The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Various light scattering methods have been used in scientific research and industrial technologies in order to determine the size distribution of particles. These methods can be divided into the following categories: 1) methods based on analysis of light scattered by a particle suspension; 2) methods based on analysis of light scattered by an individual particle. This invention is related to the latter category.
The instrumental set-up commonly known and used for flow cytometry can be applied to analysis of individual particles. The hydrofocusing system allows the measurement of scattering or/and fluorescence from individual particles with a speed varying from 10 up to 1000 particles per second. Several alternative design principles of hydrofocusing heads have been described in the literature for the purpose of measurement of various parameters from the light scattering. These methods have been reviewed by Salzman & al. in: Flow Cytometry and Sorting, Second Edition, M. R. Melamed, T. Lindmo, M. L Mendelsohn, Eds. Wiley & Sons, Inc., New York, 1990, pp. 81-107. The ordinary flow cytometer, however, does not allow absolute determination of particle parameters with the accuracy and precision required by the applications of this invention. In the ordinary flow cytometer the laser beam is focused perpendicular to the flow of particles and it measures the light scattering from microparticles in one fixed polar angle only and is not applicable for the measurement of the entire scattering pattern.
It is important to measure the entire scattering pattern for individual particles, because the angular scattering pattern completely characterises properties of the particle. In investigation of morphological characteristics of particles (size, refractive index, shape, etc.), scattering pattern works like a finger-print. Various parameters including angular locations of maxima and minima, maxima to minima ratios etc. can be determined from the angular scattering pattern and used for calculation of the morphological parameters.
In following we review the methodology known in the literature applicable for measurement of the morphological parameters as referred to above.
An empirical solution for the inverse-scattering problem of individual particles is provided by the optical strip-map technique introduced by G. M. Quist and P. J. Wyatt, (Empirical solution to the inverse-scattering problem by the optical strip-map technique, J. Opt. Soc. Am. A2, pp. 1979-1986 (1985)). This technique allows the determination of particle parameters from analysis of the intensity of the light scattered in fixed angles.
The volume distribution of red blood cells was determined by D. H. Tysko, M. H. Metz, E. A. Epstein, A. Grinbaum, "Flowcytometric light scattering measurement of red blood cell volume and haemoglobin concentration", Appl. Opt. 24, 1355-1365 (1985)). as they measured the light scattered into two angular intervals. Two photo diodes detected the scattering light collimated at polar angles from 2.5.degree. to 5.degree. and from 6.degree. to 9.degree., and at azimuthal angles from 0.degree. to 360.degree. for both detectors. Drops of heptane, nonane, and dodecane in water was used for calibration.
The Mie scattering model of marine particles was developed and tested by S. G. Ackleson and R. W. Spinard (Size and refractive index of individual marine particulates: a flow cytometric approach, Appl. Opt. 27, 1270-1277 (1988)), who used a conventional flow cytometer and well-defined polystyrene particles. The model was used for simultaneous measurements of near-forward light scatter and side scatter that yielded particle sizes within the range of d=1-10 .mu.m and refractive indexes within the range of n=1.35-1.47. The size distribution of marine particles in clear ocean waters was analyzed.
Milk fat and latex particle size distributions were analyzed by K. Takeda, Y. Ito, and C. Munakata (Simultaneous measurement of size and refractive index of a fine particle in flowing liquid, Meas. Sci. Technol. 3, 27-32 (1992)). In this study, light intensities at two different wavelengths (441.6 and 632.8 nm) and scattering in both forward and sideways directions were measured. In order to determine rapidly the size and the refractive index of any given particle, the inverse problem of Mie scattering was solved using the optical strip-map technique.
Relation of particle sizes and refractive indices to locations of minima was investigated by M. Kerker (In: The Scattering of Light and Other Electromagnetic Radiation. Academic, New York, 1969) and H. C. van de Hulst (In: Light Scattering by Small Particles, Wiley, New York, 1957). However, determining of particle parameters from the scattering data (an inverse problem of scattering) still remained as a vital problem.
An approach for the measurement of the scattering utilizing the motion of a particle in the flow of carrier liquid was described by Loken et al. (Cell discrimination by multiangle light scattering, Histochem. Cytochem. 24, 1976, 284-291).
An optical system recorded the scattering intensity versus time, which expresses the intensity of single particle scattering versus scattering angle for polar angles from 1.0.degree. to 49.0.degree.. To correct the measured scattering function for variations of the illumination intensity and for collection aperture as a function of particle position, the transfer function was determined empirically by introducing fluorescent particles into the flow.
Ludlow and Kaye (A scanning diffractometer for the rapid analysis of microparticles and biological cells, Colloid Interface Sci. 69,1979, 571-589) used a scanning diffractometer with a single photomultiplier to measure the scattering of polystyrene particles and spores. A rotating disk and 174 optical light-guides allowed the measurement of scattering intensities of single particles at polar angles from 3.degree. to 177.degree. in 2.8 ms. The size distributions and mean refractive indices were determined by comparison with the Mie scattering characteristics for homogeneous spheres.
A differential light scattering photometer described by Bartholdi et al. (Differential light scattering photometer for rapid analysis of single particles in flow, Applied Optics 19, 1573-1584 (1980)) allows momentary measurement of scattering from single particles. A ellipsoidal reflector reflects emitted scattering in polar angles from 2.5.degree. to 177.5.degree. and azimuthal angles from 0.degree. to 360.degree. onto a circular array of 60 photo diodes. This optical system performs fast measurement of the light scattering and fast analysis of single particles.